Method and System for Measuring the Pharmacokinetics of Liposomal Curcumin and its Metabolite Tetrahydrocurcumin

ABSTRACT

The present invention includes a stabilized curcumin composition. The composition includes a curcumin composition and a phosphate composition, wherein the phosphate composition is non-buffering and is provided in an amount sufficient to stabilize and/or prevent the degradation of curcumin and/or a curcuminoid in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/918,112 filed Jun. 14, 2013, which is a non-provisional applicationof U.S. Provisional Application Ser. No. 61/659,660, filed Jun. 14,2012, the entire contents of which are incorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

None.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to compositions and methods forstabilizing curcumin and tetrahydrocurcumin (THC) and in particular, tocompositions and methods for stabilizing curcumin and THC in plasma andbile against degradation occurring during analytical processes bylowering the pH with phosphoric acid.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with methods of stabilizing curcumin and THC in plasma andbile against degradation occurring during analytical processes. Curcuminis the major yellow pigment of turmeric, derived from the rhizome of theherb Curcuma longa Linn, and has traditionally been used as a treatmentfor inflammation, skin wounds, and tumors. In addition, preclinicalanimal models, curcumin has shown cancer chemo preventive,antineoplastic and anti-inflammatory properties. Curcumin[1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] has thestructure below:

Curcumin acts as a scavenger of hydroxyl radical, superoxide anion andsinglet oxygen and other oxygen species. Curcumin plays a role incellular signal induction pathways pertinent to growth, differentiationand malignant transformations, including inhibiting protein kinases,c-Jun/AP-1 activation, prostaglandin biosynthesis, and may play a rolein the activation of the transcription factor NF-κB. However, it hasbeen thought that the bioavailability of curcumin in animals remains lowwith a poor bioavailability which may be related to its inadequateabsorption and fast metabolism. Indirect evidence suggests that curcuminis metabolized in the intestinal tract where curcumin undergoesmetabolic O-conjugation to curcumin glucuronide and curcumin sulfate andbioreduction to THC, hexahydrocurcumin and hexahydrocurcuminol. Much ofthis is confirmed through examination and analysis of curcumin presentin samples (e.g., tissue extracts) before and after treatment. Instudies it has been shown that perorally administered curcumin has poorbioavailability and only low or non-measurable blood levels wereobserved. Others have administered piperine along with curcumin toenhance the bioavailability of curcumin; however, the level ofenhancement was only modest and no curcumin could be detected after 3hours even when supplemented with piperine.

U.S. Pat. No. 8,153,172, entitled “Composition to Enhance theBioavailability of Curcumin,” discloses a composition having acurcuminoid and an essential oil of turmeric. A composition having acurcuminoid and an essential oil of turmeric, wherein the essential oilis present in an amount sufficient to cause an enhancement ofbioavailability of curcumin when the composition is administered to ahuman as compared to bioavailability of curcumin obtained uponadministration of a composition prepared without adding essential oil tothe curcuminoid. A method to prepare a composition having a curcuminoidand an essential oil of turmeric.

U.S. Pat. No. 7,067,159, entitled “Methods for Treating Prostate Cancerwith Herbal Compositions,” discloses methods for treating prostatecancer, comprising administration of a composition comprisingtherapeutically effective amounts of supercritical extracts of rosemary,turmeric, oregano and ginger; and therapeutically effective amounts ofhydroalcoholic extracts of holy basil, ginger, turmeric, Scutellariabaicalensis, rosemary, green tea, huzhang, Chinese goldthread, andbarberry.

U.S. Pat. No. 7,060,733, entitled “Methods for Treating Pancreatitiswith Curcumin Compounds and Inhibitors of Reactive Oxygen Species,”discloses methods of treating, preventing, modulating, attenuating, orinhibiting a disease or a disorder associated with inflammation relatedto NF-κB activation in a subject which comprises administering to thesubject at least one curcumin compound. Also disclosed are combinationtherapies comprising the administration of at least one curcumincompound and at least one ROS inhibitor. Pharmaceutical compositions andkits are also disclosed.

U.S. Pat. No. 5,679,864, entitled “Process for the Synthesis ofCurcumin-Related Compounds,” discloses a process for the synthesis ofcurcumin and curcumin-related compounds by reacting the enol form of a2,4-diketone with a monocarbocyclic aldehyde in the presence of anorganic amine catalyst. The reactants are dissolved in a highly polar,aprotic organic solvent. The curcumin-related product is recovered incrystalline form by precipitation from the reaction mass and solventrecrystallization.

SUMMARY OF THE INVENTION

The present invention provides a method of stabilizing curcumin andtetrahydrocurcumin in the plasma and bile against degradation occurringduring analytical processes by lowering the pH with phosphoric acid. Oneembodiment of the present invention provides a method of determining acurcumin level in a biological sample by providing a biological samplecomprising a curcuminoid composition and adding a strong acid, e.g., aphosphate composition, to the sample, wherein the phosphate compositionis non-buffering and detecting the amount of curcuminoid in the sample,wherein the non-buffering strong acid reduces the degradation of thecurcuminoid in the sample. The biological sample may be an in vitrosample and include an aqueous sample, a supernatant sample, a tearssample, a sputum sample, a blood sample or a bile sample. Thecurcuminoid composition may include curcumin and analogues andderivatives selected from curcumin; tetrahydrocurcumin;hexahydrocurcumin and hexahydrocurcuminol; curcumin glucuronide; andcurcumin sulfate and the phosphate composition may include a phosphoricacid; an orthophosphoric acid; a phosphate salt; or a Na-phosphate. Thecurcuminoid composition may include a liposome, a phospholipid or apolymer composition to form an encapsulated curcuminoid composition andthe liposome, the phospholipid or the polymer composition is selectedfrom the group consisting of phosphatidylcholine (lecithin),lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine,phosphatidylinositol, sphingomyelin, phosphatidylethanolamine(cephalin), cardiolipin, phosphatidic acid, cerebrosides,dicetylphosphate, phosphatidylcholine, anddipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate; or wherein the polymer composition is selectedfrom the group consisting of polyesters, polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyamides, polyurethanes,polyesteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoesters,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), copolymers, terpolymers, and combinations or mixturesthereof and have a size of about 10-900 nm.

One embodiment of the present invention provides a stabilized curcumincomposition. The composition includes a curcumin composition and aphosphate composition, wherein the phosphate composition isnon-buffering, wherein and wherein the non-buffering strong acid reducesthe degradation of the curcuminoid in the sample. As a result thephosphate composition does not include an aqueous solution consisting ofa mixture of a weak acid and its conjugate base or a weak base and itsconjugate acid. The curcumin composition may be a curcumin composition,a modified curcumin composition or a product of a curcumin degradation,for example, the curcumin composition may be selected from curcumin;tetrahydrocurcumin; hexahydrocurcuminol; curcumin glucuronide; curcuminsulfate or other related products. In addition the curcumin compositionmay be a mixture of the 2 or more modified curcumin compositions, aproduct of a curcumin degradation, modified curcumin or syntheticcurcumin compositions. The phosphate composition is a phosphatecontaining composition that is non-buffering and as a result is notmixture of a weak acid and its conjugate base or a weak base and itsconjugate acid, e.g., not a PBS. In one embodiment of the presentinvention, the phosphate composition is a phosphoric acid. In otherembodiments the phosphate composition can be an orthophosphoric acid; aphosphate salt; a Na-phosphate; a K-phosphate; or other counter ionphosphate. In other embodiments the phosphate composition can be amixture of phosphate compositions as long as the final composition isnot a buffer, i.e., not mixture of a weak acid and its conjugate base ora weak base and its conjugate acid. The stabilized curcumin can furthercomprising a liposome to form a liposomal curcumin composition and canbe in any common dosage form known to the skilled artisan includinginfusion nanoparticle, tablet, capsule, liquid and the like.

One embodiment of the present invention includes a method of analyzing acurcumin sample by providing a sample comprising a curcumin compositionand adding a phosphate composition to the sample, wherein the phosphatecomposition is non-buffering and at least one of stabilizes or reducedthe degradation of the curcumin in the sample. The method can theninclude the step of analyzing at least one property of the sample and insome cases the sample is an aqueous sample, a blood sample or a bilesample. As a result the phosphate composition does not include anaqueous solution consisting of a mixture of a weak acid and itsconjugate base or a weak base and its conjugate acid. The curcumincomposition may be a curcumin composition, a modified curcumincomposition or a product of a curcumin degradation, for example, thecurcumin composition may be selected from curcumin; tetrahydrocurcumin;hexahydrocurcuminol; curcumin glucuronide; curcumin sulfate or otherrelated products. In addition the curcumin composition may be a mixtureof the 2 or more modified curcumin compositions, a product of a curcumindegradation, modified curcumin or synthetic curcumin compositions. Thephosphate composition is a phosphate containing composition that isnon-buffering and as a result is not mixture of a weak acid and itsconjugate base or a weak base and its conjugate acid, e.g., not a PBS.In one embodiment of the present invention, the phosphate composition isa phosphoric acid. In other embodiments the phosphate composition can bean orthophosphoric acid; a phosphate salt; a Na-phosphate; aK-phosphate; or other counter ion phosphate. In other embodiments, thephosphate composition can be a mixture of phosphate compositions as longas the final composition is not a buffer, i.e., not mixture of a weakacid and its conjugate base or a weak base and its conjugate acid.

One embodiment of the present invention provides a method of stabilizinga curcumin or tetrahydrocurcumin sample by providing a sample comprisingcurcumin composition and adding a phosphate composition to the sample,wherein the phosphate composition is non-buffering and at least one ofstabilizes or reduced the degradation of the curcumin in the sample. Asa result, the phosphate composition does not include an aqueous solutionconsisting of a mixture of a weak acid and its conjugate base or a weakbase and its conjugate acid. The curcumin composition may be a curcumincomposition, a modified curcumin composition or a product of a curcumindegradation; for example, the curcumin composition may be selected fromcurcumin, tetrahydrocurcumin, hexahydrocurcuminol, curcumin glucuronide,curcumin sulfate or other related products. In addition, the curcumincomposition may be a mixture of the 2 or more modified curcumincompositions, a product of a curcumin degradation, modified curcumin orsynthetic curcumin compositions. The phosphate composition is aphosphate containing composition that is non-buffering and as a resultis not a mixture of a weak acid and its conjugate base or a weak baseand its conjugate acid, e.g., not a PBS. In one embodiment of thepresent invention, the phosphate composition is a phosphoric acid. Inother embodiments the phosphate composition can be an orthophosphoricacid, a phosphate salt, a Na-phosphate, a K-phosphate, or other counterion phosphate. In other embodiments, the phosphate composition can be amixture of phosphate compositions as long as the final composition isnot a buffer, i.e., not a mixture of a weak acid and its conjugate baseor a weak base and its conjugate acid.

One embodiment of the present invention provides a curcumin diagnostickit including a non-buffering phosphate composition and a set ofinstructions for stabilizing a curcumin sample using the non-bufferingphosphate composition, wherein the amount of a non-buffering phosphatecomposition sufficient to stabilize a curcuminoid in a biologicalsample, and wherein the non-buffering phosphate composition comprises aphosphoric acid; an orthophosphoric acid; a phosphate salt; or aNa-phosphate to stabilize Curcumin; tetrahydrocurcumin;hexahydrocurcumin and hexahydrocurcuminol; curcumin glucuronide; andcurcumin sulfate. As a result the phosphate composition does not includean aqueous solution consisting of a mixture of a weak acid and itsconjugate base or a weak base and its conjugate acid.

Another embodiment of the present invention provides a method ofstabilizing a curcumin composition in plasma sample or a bile sampleagainst degradation during an analytical processes by providing a samplecomprising a curcumin composition, wherein the sample is a bile sampleor a blood sample and the curcumin composition is selected fromCurcumin; tetrahydrocurcumin; hexahydrocurcumin and hexahydrocurcuminol;curcumin glucuronide; and curcumin sulfate and adding a phosphatecomposition to the sample, wherein the amount of a non-bufferingphosphate composition is sufficient to stabilize a curcuminoid in abiological sample. As a result the phosphate composition does notinclude an aqueous solution consisting of a mixture of a weak acid andits conjugate base or a weak base and its conjugate acid. The curcumincomposition may be a curcumin composition, a modified curcumincomposition or a product of a curcumin degradation, for example, thecurcumin composition may be selected from curcumin; tetrahydrocurcumin;hexahydrocurcuminol; curcumin glucuronide; curcumin sulfate or otherrelated products. In addition the curcumin composition may be a mixtureof the 2 or more modified curcumin compositions, a product of a curcumindegradation, modified curcumin or synthetic curcumin compositions. Thephosphate composition is a phosphate containing composition that isnon-buffering and as a result is not mixture of a weak acid and itsconjugate base or a weak base and its conjugate acid, e.g., not a PBS.In one embodiment of the present invention, the phosphate composition isa phosphoric acid. In other embodiments the phosphate composition can bean orthophosphoric acid; a phosphate salt; a Na-phosphate; aK-phosphate; or other counter ion phosphate. In other embodiments thephosphate composition can be a mixture of phosphate compositions as longas the final composition is not a buffer, i.e., not mixture of a weakacid and its conjugate base or a weak base and its conjugate acid. Thestabilized curcumin can further comprising a liposome to form aliposomal curcumin composition and can be in any common dosage formknown to the skilled artisan including infusion nanoparticle, tablet,capsule, liquid and the like.

Another embodiment of the present invention provides a method ofperforming a clinical trial to evaluate a candidate drug comprising acurcumin or curcuminoid believed to be useful in treating a medicalcondition, the method comprising: (a) obtaining a first tissue samplesprior to providing the candidate substance from tissue suspected from aset of patients; (b) administering the candidate drug to a first subsetof the patients, and a placebo to a second subset of the patients; (c)repeating step (a) after the administration of the candidate drug or theplacebo; and (d) obtaining a second tissue sample from the first andsecond set of patients and stabilizing the curcumin or curcuminoids inthe second tissue samples by adding an effective amount of anon-buffering phosphate; and (e) determining of there is a statisticallysignificant difference in the amount of curcumin or curcuminoids in thesecond tissue samples between the first and second subset of patients,wherein a statistically significant reduction indicates that thecandidate drug is useful in treating said disease state.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A-1D are graphs of the plasma levels of curcumin and THC as afunction of time after infusion.

FIGS. 2A-2D are graphs of the plasma, bile and urine curcumin levels asa function of time after infusion.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The term “liposome” refers to a capsule wherein the wall or membranethereof is formed of lipids, especially phospholipid, with the optionaladdition therewith of a sterol, especially cholesterol.

As used herein, the term “in vivo” refers to being inside the body. Theterm “in vitro” used as used in the present application is to beunderstood as indicating an operation carried out in a non-livingsystem.

The terms “effective amount” or “therapeutically effective amount”described herein means the amount of the subject compound that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The term “pharmaceutically acceptable” as used herein to describe acarrier, diluent or excipient must be compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

The term “curcumin” as used herein to describe (i) curcumin derivativesor combinations thereof dissolved or dispersed in an aqueous or anon-aqueous solvent with one or more optional related co-factors,proteins, antibodies, pain medications, and other pharmaceuticallyactive agents dissolved, dispersed or suspended in the solvent, (ii) asuitable aqueous or non-aqueous dispersion medium, wherein the one ormore spherical liposomes are dispersed in the dispersion medium, and(iii) one or more optional excipients, diluents, extended or controlledrelease agents, lubricants, preservatives or any combinations thereof.

The present invention provides the stabilization of curcumin and/or THCin plasma; however, the stabilization is more complicated than theacidification of plasma with H₃PO₄. For example, in Phase 1, humanplasma samples were stabilized with the addition of Na-phosphate,however, the bench stability of both curcumin and THC was minimal and nocurcumin and/or THC were detected after the samples sat on the bench atroom temperature for a couple of hours. The addition of Na-phosphatestabilized the plasma, although not as efficient as with H₃PO₄. As aresult, one embodiment of the present invention provides the stabilityof curcumin and THC in plasma by the addition of Na-phosphate. Anotherembodiment of the present invention provides the stability of curcuminand THC in plasma by the addition of O-Phosphoric acid. Anotherembodiment of the present invention provides the stability of curcuminand THC in plasma by the addition of Na-phosphate and O-Phosphoric acid.O-Phosphoric acid can be more easily incorporated in the plasma or bodyfluids. The phosphate molecule is essential for stabilizing the curcuminand THC, both the sodium phosphate and phosphoric acid can be used. Incontrast, a phosphate buffer does not stabilize curcumin and THC inplasma. The phosphate buffer is not specific to which salt it is madefrom and can be made with either sodium or potassium phosphate(monobasic or dibasic) whereas sodium phosphate monobasic is specific asis orthophosphoric acid. The main purpose of adding thephosphate/phosphoric acid is to stabilize the curcumin and THC in theplasma. As we have seen the presence of the phosphoric acid in theplasma shows a higher concentration for the analytes. This stabilisationprocess may as well be achieved by using other phosphate salts known tothe skilled artisan. The addition of H₃PO₄ leads to higher curcuminconcentrations because of the shift in pH confirmation. For example, thepH of EDTA plasma is 7.95, which is a little higher than the pH ofplasma without EDTA. After the addition of 50 μl % H₃PO₄ to 950 mlplasma the pH was 4.4. As curcumin is stable at pH values below 5.5 theaddition of H₃PO₄ stabilizes curcumin in the plasma samples. Otherembodiments may use other acidification agents (e.g. ACD, acidiccitrate, etc.,) and may also use anticoagulants. In addition, the urinetends to be more acidic with a pH of between 5 and 7; however, nostabilization was observed with H₃PO₄.

The present invention provides a method of stabilizing curcumin and THCin the plasma and bile against degradation occurring during analyticalprocesses by lowering the pH with phosphoric acid. In one study of 4dogs, 2 males and 2 females were infused with 10 mg/kg liposomalcurcumin (LIPOCURC™) over 2 hours, and another 4 dogs, 2 males and 2females were infused with 10 mg/kg liposomal curcumin (LIPOCURC™) over 8hours. Plasma levels of curcumin and THC were obtained at necropsy 15minutes following the infusion. THC levels were 6.3-9.6 fold higher thancurcumin at both infusion rates suggesting a combination of a high rateof enzymatic curcumin metabolism and a comparatively slower rate ofblood THC clearance. Compared to the 8 hour infusion, the 2 hourinfusion levels of both curcumin and its metabolite THC weresignificantly higher. The plasma half lives of both compounds followingthe 2 hour infusion ranged from 0.4-0.7 hours, and was a consequence ofboth hepatic and renal clearance. However at higher plasmaconcentrations renal excretion predominates particularly with THC.Enhanced clearance rates were noted during the 8 hour infusions whichprevented achieving a steady state. These observations suggest that forhematopoietic malignancies including leukemia, lymphoma, and bone marrowmetastases, the 2 hour infusion may be advantageous based upon higherconcentration profiles, and the unstimulated clearance rates.

The parenteral administration of liposomal curcumin (LIPOCURC™) withtherapeutic intent poses several questions relating to deciding anoptimal rate of administration for patients with neoplastic diseases.Options ranging from bolus intravenous injections to constant infusionsare impacted by enzymatic metabolism, pH dependent degradation, renaland hepato-biliary excretion mechanisms. During pre-clinicaltoxicological evaluation in dogs, dose dependent hemolysis was notedfollowing brief infusions of 20 mg/kg and greater curcumin content. Tenmg/kg doses infused over 2 hours were nontoxic. This same 2 hourinfusion schedule was used in an ascending dose Phase 1 trial in normalhuman subjects where the highest intravenous dose administered (5 mg/kg)was without adverse reaction. To avoid toxicity from a too-high C_(max)we used a two hour infusion, however in view of the unknown metabolicand elimination factors in dogs we compared 2 hour and 4 fold longerinfusions (8 hours) to determine any advantages.

Plasma concentration data arising from the infusion of liposomalcurcumin (LIPOCURC™) in 8 dogs (4 females and 4 males) of the Beaglebreed were used. The results and analysis for the study are presentedfor intravenous infusion dosing of a total dose of 10 mg/kg infused overa period of either 2 or 8 hours. Plasma levels of curcumin and itsmetabolite, THC were measured at timed intervals post-dosing. Allanimals were euthanized and subject to necropsy 15 minutes post-infusionand samples of tissues, plasma, bile, and urine taken to determine, thetissue distribution and pharmacokinetics of curcumin and THC followingtwo different rates of infusion and two different analytepreservation/stabilization methods, e.g., with and/or without phosphoricacid (H₃PO₄) and the plasma pharmacokinetics, urine and bile levels ofcurcumin and THC reviewed. A summary of the treatment groups ispresented in Table 1 below.

TABLE 1 Summary of Treatment Groups Concentration of Infusion Durationof Number of Beagle Dose Curcumin Rate Infusion Dogs On Study^(a) Groups(mg/kg) (mg/mL) mL/kg/hr (hr) M F Part A, Liposomal 10 0.5 10 2 2 2Curcumin Part B, Liposomal 10 0.125 10 8 2 2 CurcuminLiposomal curcumin (LIPOCURC™) was administered to 8 Beagle dogs byintravenous infusion over two hours (Part A) or eight hours (Part B).For the 2 hour infusion, blood samples were taken at predose and 0.25,0.5, 1.5 and at 2 hours during infusion and at 15 minutes post-infusion.For the 8 hour infusion, blood samples were taken at predose and 0.25,0.5, 1.5, 4, 4, 6 and at 8 hours during infusion and at 15 minutespost-infusion.

For all groups, plasma curcumin and THC were determined using a methoddeveloped by the Bioanalytical Department at Nucro-Technics [1].Bioanalysis was performed on two sets of samples, one set that wastreated with phosphoric acid and one set that was not treated withphosphoric acid. Phosphoric acid was used to treat one set of samplesbased on preliminary studies indicating that phosphate increased thestability of curcumin and THC in the tissue matrix. Values that werebelow the limit of quantification were assigned a value of 0.

As there were no consistent differences between the plasma levels ofcurcumin in male dogs or female dogs, the average plasma concentrationsfrom male and female dogs were used to perform the PK analysis. Plasmaconcentration vs. time profiles were analyzed using (unless otherwisestated) the data from 4 dogs. Plasma profiles for the test articles arepresented as the mean data±SE of 4 dogs. Average plasma concentrationswere used to perform the PK analysis. Plasma concentration vs. timeprofiles were analyzed and the PK parameters estimated using WinNonlinVersion 5.2.1 employing the intravenous infusion model with first orderelimination. Unless stated otherwise, the plasma concentration-timeprofiles for the test articles are presented as the mean data±SE of 4dogs.

FIGS. 1A-1D are graphs of the plasma levels of curcumin as a function oftime after infusion. FIG. 1A is a graph of the plasma level of curcuminfollowing a 2 hour infusion of 5 mg/kg/hr of curcumin. FIG. 1B is agraph of the plasma level of curcumin following an 8 hour infusion of1.25 mg/kg curcumin. FIG. 1C is a graph of the plasma level of THCfollowing a 2 hour infusion of 5 mg/kg/hr curcumin. FIG. 1D is a graphof the plasma level of THC following an 8 hour infusion of 1.25 mg/kg/hrcurcumin. Values are presented as the mean±standard error of 4 dogs.

The plasma levels and AUC of curcumin and THC following either 2 hours(high rate) or 8 hours (low rate) infusion were clearly higher in thepresence of phosphoric acid (Tables 2 and 3), suggesting that phosphoricacid increased the stability of curcumin and THC in plasma samples.

Table 2 below is a table of the AUC of plasma concentration vs. time forcurcumin and THC upon bioanalysis in the presence and absence ofphosphoric acid. Phosphoric acid was added to the plasma samples in theform of phosphoric acid; C_(max) represents the observed value and AUCis the area under the curve to 15 minutes post-infusion calculated usingthe linear trapezoidal rule.

AUC (ng/mL*hr) C_(max) (ng/mL) Infusion Time Curcumin THC Curcumin THC 2hr 65 1318 46 891 2 hr + phosphate 394 3797 320 2983 8 hr 52 411 15 77 8hr + Phosphate 187 1171 66 293

Table 3 below is a table of the plasma concentration vs. time forcurcumin and THC upon bioanalysis in the presence and absence ofphosphoric acid.

Infusion Rate [Plasma], ng/mL [Plasma + PO₄], ng/mL and Time CurcuminTHC Curcumin THC  5 mg/kg/hr Pre-Dose  0 ± 0  0 ± 0 0 ± 0  0 ± 0 15 min20 ± 2 483 ± 50 8 ± 3 284 ± 89 30 min 25 ± 5 566 ± 77 77 ± 39 1116 ± 318 90 min² 36 ± 3  891 ± 238 319 ± 91  2352 ± 441 2 hr  46 ± 23 454 ± 79257 ± 46  2983 ± 852 1.25 mg/kg/hr Pre-Dose  0 ± 0  0 ± 0 0 ± 0  0 ± 015 min  0 ± 0  72 ± 15 13 ± 8   59 ± 24 30 min  5 ± 2  63 ± 15 32 ± 12121 ± 28 90 min  9 ± 1  64 ± 14 65 ± 16 293 ± 73 2 hr  3 ± 1  68 ± 11 38± 14 226 ± 64 4 hr 12 ± 1 77 ± 8 30 ± 28  193 ± 127 6 hr  0 ± 0  0 ± 0 0± 0  64 ± 10 8 hr 15 ± 4  67 ± 29 6 ± 2  62 ± 26Values are presented as the mean±SE of 4 values.This was also the case for bile, but less so, while for urine the impactof the addition of phosphoric acid was variable.

FIGS. 2A-2D are graphs of the plasma, bile, and urine curcuminpost-infusion levels as a function of time after infusion plasma, bile,and urine levels. FIG. 2A is a graph of curcumin levels following a 2hour infusion of 5 mg/kg/hr curcumin. FIG. 2B is a graph of curcuminlevels following an 8 hour infusion of 1.25 mg/kg curcumin. FIG. 2C is agraph of THC levels following a 2 hour infusion of 5 mg/kg/hr curcumin.FIG. 2D is a graph of THC levels following an 8 hour infusion of 1.25mg/kg/hr curcumin. Table 3 shows THC in the absence of phosphoric acid,the value is presented as the mean±SE of three determinations, otherwiseall values are presented as the mean±standard error of 4 dogs.

Equivocal data for the bioanalysis of curcumin in the plasma of rats hasbeen observed in the literature following oral administration of highdoses [2]. Detection methods rather than plasma stability werespeculated as the reason for the discrepancy, however, it appears thatplasma/tissue stability would also be an issue in the bioanalysis ofcurcumin. One embodiment of the present invention provides thequantification of curcumin and THC stabilized by phosphoric acid inplasma, bile, and urine samples.

Upon a 2 hour infusion of curcumin at 5 mg/kg/hr (total dose 10 mg/kg),the plasma levels of curcumin rose to attain a maximum concentration of320 ng/mL by 1.5 hours and then began to stabilize/fall during theinfusion. Upon cessation of the infusion, there was a rapid drop inplasma concentrations of curcumin from 257 ng/mL to 65 ng/mL in 15minutes. THC had a similar concentration-time profile. For the 8 hourinfusion of curcumin at a rate of 1.25 mg/kg/hr (total dose 10 mg/kg),peak plasma concentrations of 187 ng/mL were also reached by 1.5 hoursand then began to fall during the infusion period and thus, steady-statelevels were not achieved; a similar concentration-time profile was alsoobserved for THC. The ratio of THC to curcumin based on AUC was 9.6 forthe 2 hour infusion and 6.3 for the 8 hour infusion. The drop in plasmalevels of both curcumin and its metabolite, THC, upon the 8 hourinfusion suggests that infusion of curcumin may activate or enhance itsown elimination.

Computer assisted pharmacokinetic analysis of the plasma concentrationdata was only shown for the 2 hour infusion. The estimated PK parametersfor curcumin and THC are shown in Table 4, while the C_(max observed)and calculated AUC are shown in Table 2.

Table 4 below illustrates the estimated PK parameters of curcumin andTHC. For a 2 hour intravenous infusion at a dose rate of 2 mg/kg/hr;total dose 10 mg/kg. The estimated PK parameters were determined byfitting the data to a first-order elimination continuous intravenousinfusion model.

Parameter Units Curcumin THC AUC ng*hr/mL 485 5185 C_(max) ng/mL 2332429 t_(1/2(e)) ¹ hr 0.4 0.5 Ke¹ hr⁻¹ 1.6 1.4 MRT¹ hr 0.6 0.7 CL L/hr/kg20.6 Vss L/kg 12.7

The rapid decrease in plasma concentration of curcumin is consistentwith short t_(1/2(e)) and MRT values of 0.4 and 0.6 hours respectivelyas a result of a high clearance of 20.6 L/kg/hr from a volume ofdistribution of 12.7 L/kg. The fitted C_(max) and AUC values of 233ng/mL and 485 ng*hr/mL are close to the observed C_(max) of 320 ng/mLand calculated AUC of 394 ng*hr/mL. THC had estimated t_(1/2(e)) and MRTvalues close to those of curcumin with the estimated values being 0.5and 0.7 hours, respectively with C_(max) and AUC values of 2429 ng/mLand 5185 ng*hr/mL, compared to the observed values of 2983 ng/mL and3797 ng*hr/mL. The observed C_(max) values for curcumin at infusion doserates of 1.25 and 5.0 mg/kg/hr were close to being dose-proportional tothe dosing rate, with dosing rate normalized C_(max) values(C_(max)/Dosing rate in mg/kg/hr) of 64 and 53 ng/mL observed for the 2and 8 hour infusions. The AUDs and infusion dose rate normalized AUDs upto 2 hours for the high and the low infusion rates were 354 and 82ng*hr/mL and 59 and 66 ng*hr/mL, respectively, also consistent withdose-proportionality.

Measurement of the levels of curcumin and THC in the plasma, urine, andbile provide additional information concerning the disposition ofcurcumin (FIG. 2A-2D; Table 5 below). For bile, the levels of curcuminand THC were somewhat higher in female dogs compared to the male dogs.At both the high and low infusion rate of 1.25 mg/kg/hr, curcumin wasfound at higher concentrations in the urine and bile compared to plasma.At the low infusion rate, the urine and bile to plasma concentrationratios were 10 and 32, respectfully while at the higher infusion rate,the values observed were 44 and 16, respectfully.

Table 5 illustrates plasma, urine, and bile levels of curcumin and THC15 minutes, 2 hours and 8 hours post infusion.

Mattrix 2 hour 8 hour 2 hour 8 hour [Curcumin], ng/mL [Curcumin +H₃PO₄], ng/mL Plasma 0 ± 0 0 ± 0 65 ± 28  14 ± 141 Urine 3657 ± 932  369± 247 2842 ± 170  148 ± 87  Bile 590 ± 224 292 ± 83  1028 ± 539  449 ±96  [THC], ng/mL) [THC + H₃PO₄], (ng/mL) Plasma 38 ± 4  20 ± 42 1167 ±379  142 ± 122 Urine 6417 ± 1450 2451 ± 84  3587 ± 1083 621 ± 206 Bile187 ± 74  84 ± 12 391 ± 197 168 ± 53 Unless indicated otherwise, values are the mean±SE of 4 determinations.Three values were 0 and one value was 58 ng/mL. Mean±SE of 3determinations.

The liver and the kidney can eliminate curcumin from the plasma and athigher plasma concentrations the kidney can excrete more curcumin whilebiliary excretion is approaching saturation. This is consistent withstudies in rats where tissue disposition studies of intravenouslyadministered curcumin demonstrated the highest exposure in the liver andkidney [3]. Modulation of renal transporters may play an important rolein the enhancement of the elimination of curcumin previously mentioned.For THC the urine to plasma concentration ratios were higher than thebile to plasma concentration ratios, both at the low and high infusionrates, with values of 3.1 and 4.4 compared to 0.3 and 1.2, respectively.This is consistent with metabolism of curcumin to THC by the hepatic andextra-hepatic tissues, accumulation of THC in the plasma and excretionvia the urine.

These data demonstrate drug stability, dose, and schedule ofadministration represent important and malleable components of curcuminclinical therapeutics. Tissue phenotype, metabolism, excretion routes,transport mechanisms and distribution are important but less subject tomodification. Of these parameters curcumin degradation prior to andduring analytic procedures is critically important and contributes tothe variences and validity of plasma levels reported in animal studiesof oral and parenteral curcumin administration. The high susceptibilityto ambient light and pH of curcumin was resolved by the addition ofphosphoric acid to stabilize curcumin prior to analytical processing.

Another factor contributing to misinformation regarding curcumin bloodlevels in animal models is the effect of metabolic activity. Curcumincan be released as free curcumin from any of the delivery vehicles, anddistributes mainly to circulating and tissue lipids because of lowaqueous solubility or is metabolized to a number of secondary compoundsvia conjugation with glucuronides or sulfates, or reduced todihydrocurcumin, THC and octahydrocurcumin. Although the specific andcollective biological activity of these metabolites in animal models hasnot been published. The predominant reduced metabolite is THC and has asimilar biological activity to curcumin and can be converted byNADH-dependent dihydrocurcumin by intestinal E. Coli. THC can also beconverted from curcumin via a specific enzyme reductase, which has amolecular mass of 82 KDa and consists of two identical subunits with arestricted substrate spectrum, preferentially acting on curcumin. Itsmechanism of action on curcumin is rendered in two steps (i.e., twoenzyme reactions). The first is a NADPH-dependent reduction to anintermediate dihydrocurcumin and the second is NADPH-dependentcurcumin/dihydrocurcumin reductase to THC. The enzyme is part of themedium chain dehydrogenase-reductase superfamily, and its presenceraises intriguing issues of enzyme origins and distribution. It is foundin the blood of mice following intraperitoneal administration ofcurcumin, and it is assumed that the enzyme is also present in humanblood and tissues: particularly the liver in humans. It is also found ina particular strain of human origin intestinal E. coli: K-12 substr.MG1655 version 15.1. While there are no published studies reporting onlevels of this reducing enzyme in animal models, the significantpresence of THC in the plasma of the dogs strongly suggests the presenceof the enzyme in tissues and blood.

The addition of phosphoric acid to plasma and bile samples in dogsprevented the degradation of curcumin and THC, which raises issues ofvalidity of published data on curcumin distribution and excretion.Infusion of liposomal curcumin (LIPOCURC™) in dogs at two differentinfusion rates resulted in higher plasma levels of curcumin and THC witha 2 hour infusion compared to an 8 hour infusion. The C_(max) andAUC_(2 hr) normalized to the infusion dose rate were proportional. Theplasma levels of THC were higher than curcumin with the ratio of plasmaTHC to curcumin ranging from 6.3-9.6. These data emphasize the putativepresence of a curcumin reducing enzyme in blood or tissues.

Analysis of the 2 hour curcumin infusion data provided estimates of theplasma t_(1/2(e)) and the mean residence times (MRT) which were short,ranging from 0.4-0.7 hours. The short plasma t_(1/2(e)) and MRT arelikely a consequence of the clearance of curcumin by both hepatic andrenal routes. Clearances of curcumin and THC over 8 hours infusion areaugmented, preventing attainment of a steady-state. The mechanism maypotentially be through modulation of renal transporters. The presentinvention provides a 2 hour infusion of curcumin, THC or curcumin andTHC would be preferable for liquid malignancies while the 8 hourinfusion of curcumin, THC or curcumin and THC for solid tumors in theabsence of tumor cell/tissue data.

In addition the present invention may be administered intravenously atherapeutically effective amount of a pharmaceutical compositioncurcumin, curcumin analogues, curcumin derivatives or combinationsthereof dissolved or dispersed in a suitable aqueous or non-aqueousmedium, wherein the curcumin is enclosed in one or more sphericalliposomes or is conjugated to one or more biodegradable polymers. Inanother aspect the liposomes comprise a lipid or a phospholipid wall,wherein the lipids or the phospholipids are selected from the groupconsisting of phosphatidylcholine (lecithin), lysolecithin,lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol,sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine,and dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate. In a specific aspect the one or more liposomeshave a size of about 100 nm. In another aspect the therapeuticallyeffective amount comprises 50 nM/kg of body weight of the subject. Inyet another aspect the pharmaceutical composition is optionallyadministered along with related co-factors, proteins, antibodies, painmedications, and other pharmaceutically active agents. In another aspectof the method disclosed hereinabove the one or more pharmaceuticallyactive agents are selected from the group consisting of L-dopa,Carbidopa, benserazide, Tolcapone, dopamine agonists bromocriptine,pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine,lisuride, MAO inhibitors, selegiline, and rasagiline.

In one aspect of the composition disclosed hereinabove the one or morespherical liposome or the polymer conjugate may be dispersed in adispersion medium, wherein the dispersion medium is an aqueous ornon-aqueous dispersion medium. In related aspects the lipid or thephospholipid is selected from the group consisting ofphosphatidylcholine (lecithin), lysolecithin,lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol,sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine,and dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate and the one or more biodegradable polymers areselected from the group consisting of polyesters, polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyamides,polyurethanes, polyesteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoesters,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), copolymers, terpolymers, and combinations or mixturesthereof.

In another aspect the composition is administered intravenously,sub-cutaneously, intra-muscularly, or intra-peritoneally. In a specificaspect the one or more liposomes have a size of about 100 nm. In yetanother aspect the composition is administered intravenously.

In another aspect the present invention may include lipid or thephospholipid selected from the group consisting of phosphatidylcholine(lecithin), lysolecithin, lysophosphatidylethanol-amine,phosphatidylserine, phosphatidylinositol, sphingomyelin,phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid,cerebrosides, dicetylphosphate, phosphatidylcholine, anddipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate. In yet another aspect the composition isadministered intravenously, sub-cutaneously, intra-muscularly orintra-peritoneally. In another aspect the one or more liposomes have asize of about 100 nm. In a specific aspect the composition isadministered intravenously.

In specific aspects of the method described hereinabove the one or moreliposomes have a size of about 100 nm and the therapeutically effectiveamount comprises 50 nM/kg of body weight of the subject. In a relatedaspect the pharmaceutical composition is optionally administered alongwith related co-factors, proteins, antibodies, pain medications, andother pharmaceutically active agents, wherein the pharmaceuticallyactive agents comprise serotonin reuptake inhibitors sertraline andparoxetine.

Tissue concentration data arising from the infusion of liposomalcurcumin in 8 (4 female and 4 male) Beagle dogs were used to assemblethis report. The results and analysis are presented for 12 tissuesamples (brain cortex, hippocampus, striatum, brain stem, heart, lungs,muscle, liver, kidney, pancreas, intestinal wall and urinary bladder)following the termination of intravenous infusion at a total dose of 10mg/kg infused over a period of either 2 or 8 hours. Tissue levels ofcurcumin and its metabolite, tetrahydrocurcumin (THC) were measured inanimals that were killed and subject to necropsy 15 minutespost-infusion to determine the tissue distribution and pharmacokineticsof curcumin and THC following two different rates of infusion and twodifferent analyte preservation/stabilization methods (with and withoutH₃PO₄).

The test article will be administered to 8 Beagle dogs by intravenousinfusion over two hours (Part A) or eight hours (Part B) as shown inTable 6.

TABLE 6 Summary of Treatment Groups Concentration of Infusion Durationof Number of Beagle Dose Curcumin Rate Infusion Dogs On Study^(a) Groups(mg/kg) (mg/mL) mL/kg/hr (hr) M F 1. Part A, Liposomal 10 0.5 10 2 2 2Curcumin 2. Part B, Liposomal 10 0.125 10 8 2 2 Curcumin

Fifteen minutes following either the 2 hour or 8 hour infusion, blood,urine and bile samples were taken, prior to the dogs being necropsiedand organs removed for the isolation of tissues. Multiple samples oftissue weighing approximately 1 gram were removed and snap frozen in thepresence or absence of phosphoric acid (H₃PO₄). For all tissue samples,the levels of curcumin and THC were determined using a method developedby the Bioanalytical Department at Nucro-Technics. Phosphoric acid wasused to treat one set of samples based on preliminary studies indicatingthat phosphate increased the stability of curcumin and THC in the tissuematrix. Values that were below the limit of quantification were assigneda value of 0. As there were no consistent differences between the tissuelevels of curcumin in males and female dogs, the average plasmaconcentrations from male and female dogs was used to assess the tissuedistribution results. Tissue distribution data was analyzed using,unless stated, the data from 4 dogs and are presented as themean±standard error (S.E.).

The distribution of curcumin and THC in tissues is illustrated in Tables7-11. In general, curcumin and THC were widely distributed amongst the12 tissues assessed. While in plasma the addition of phosphoric acid hada clear stabilizing effect on both the levels of curcumin and THC, theeffects in tissues was less clear and to some extent tissue dependentand more evident for THC. Thus, despite the high degree of variabilityfor some tissues, for brain tissue, phosphoric acid had a clearstabilizing effects, again more prominent for THC, while in othertissues, the stabilizing effect of phosphoric acid was minor or absent(i.e. heart and kidney). These differences may arise as a consequence ofdiffering metabolic capabilities for each tissue.

TABLE 7 Tissue Distribution of Curcumin in the Presence and Absence ofH₃PO₄ following 2 hour infusions. Levels (ng/g)¹ Tissue No H₃PO₄ S.E.Plus H₃PO₄ S.E. Cortex, Brain 0.52 0.05 0.74 0.13 Hippocampus 0.09 0.000.09 0.09 Striatum 0.33 0.10 0.48 0.07 Brain Stem 0.30 0.04 0.45 0.06Heart 0.49 0.08 0.48 0.09 Lungs 86.82 24.99 22.86 2.14 Muscle 1.23 0.320.19 0.02 Liver 4.28 1.90 1.82 0.45 Kidney 1.03 0.17 0.89 0.15 Pancreas2.02 0.71 0.92 0.35 Intestinal Wall 2.97 0.98 1.14 0.26 Urinary Bladder0.60 0.07 0.69 0.12 ¹Phosphate was added to the tissue samples in theform of phosphoric acid

TABLE 8 Tissue Distribution of THC in the Presence and Absence of H₃PO₄following 2 hour infusions. Levels (ng/g)¹ Tissue No H₃PO₄ S.E. PlusH₃PO₄ S.E. Cortex, Brain 0.68 0.05 3.08 0.30 Hippocampus 0.75 0.09 6.461.82 Striatum 6.22 3.10 11.12 1.42 Brain Stem 2.34 0.34 10.62 1.30 Heart2.51 0.68 0.69 0.42 Lungs 24.99 5.11 2.14 2.67 Muscle 5.26 1.33 4.191.03 Liver 1.90 0.67 0.45 0.81 Kidney 3.06 0.63 4.25 0.61 Pancreas 2.020.71 0.92 0.35 Intestinal Wall 0.73 0.40 2.12 0.89 Urinary Bladder 0.840.20 0.87 0.34 ¹Phosphate was added to the tissue samples in the form ofphosphoric acid.

TABLE 9 Tissue Distribution of Curcumin in the Presence and Absence ofH₃PO₄ following 8 hour infusions. Levels (ng/g)¹ Tissue No H₃PO₄ S.E.Plus H₃PO₄ S.E. Cortex, Brain 0.72 0.18 0.81 0.15 Hippocampus 0.00 0.000.01 0.01 Striatum 0.15 0.02 0.49 0.08 Brain Stem 0.41 0.10 0.58 0.04Heart 0.67 0.15 0.75 0.17 Lungs 317.93 101.28 250.75 56.42 Muscle 3.251.31 0.79 0.24 Liver 39.38 13.70 28.38 10.30 Kidney 2.71 0.65 2.77 1.04Pancreas 1.88 0.62 2.84 0.76 Intestinal Wall 1.79 0.53 0.84 0.17 UrinaryBladder 3.24 1.37 2.26 0.51 ¹Phosphate was added to the tissue samplesin the form of phosphoric acid.

TABLE 10 Tissue Distribution of THC in the Presence and Absence of H₃PO₄following 8 hour infusions. Levels (ng/g)¹ Tissue No H₃PO₄ S.E. PlusH₃PO₄ S.E. Cortex, Brain 0.06 0.04 0.49 0.12 Hippocampus 0.01 0.01 1.130.35 Striatum 1.12 0.11 3.14 0.26 Brain Stem 0.83 0.08 3.02 0.37 Heart0.51 0.08 0.03 0.03 Lungs 10.81 2.50 6.36 2.13 Muscle 0.38 0.25 0.930.20 Liver 2.63 0.62 2.25 0.57 Kidney 1.32 0.18 2.04 0.32 Pancreas 0.340.19 1.34 0.52 Intestinal Wall 0.31 0.31 0.21 0.12 Urinary Bladder 1.370.42 1.11 0.41 ¹Phosphate was added to the tissue samples in the form ofphosphoric acid

TABLE 11 H₃PO₄ stabilized Tissue Partition Coefficients (Kp) forCurcumin and THC following 2 hour and 8 hour infusions. Kp[tissue]/[plasma]¹ Tissue Curcumin, 2 hr THC, 2 hr Curcumin, 8 hr THC, 8hr Cortex, Brain 0.0134 0.0006 0.0544 0.0047 Hippocampus 0.0016 0.00140.0007 0.0108 Striatum 0.0087 0.0039 0.0329 0.0300 Brain Stem 0.00810.0037 0.0389 0.0289 Heart 0.0087 0.0000 0.0503 0.0003 Lungs 0.41260.0078 16.8289 0.0609 Muscle 0.0034 0.0011 0.0530 0.0089 Liver 0.03290.0028 1.9047 0.0215 Kidney 0.0161 0.0025 0.1859 0.0195 Pancreas 0.01660.0017 0.1906 0.0128 Intestinal Wall 0.0206 0.0003 0.0564 0.0020 UrinaryBladder 0.0125 0.0014 0.1517 0.0106

¹The plasma concentrations used to calculate the tissue partitioncoefficients were an average of the plasma concentration measured at theend of the infusion period and 15 minutes post infusion and were for 2and 8 hours curcumin concentrations, 55.4 and 14.9 ng/mL, respectivelyand for 2 and 8 hours THC concentrations, 810.9 and 104.5 ng/mL,respectively.

For the purpose of consistency with the discussion of the plasma, bileand urine PK of curcumin and THC, the tissue distribution results willbe discussed for tissue levels determined in the presence of phosphoricacid. Curcumin and THC were distributed in all of the tissuesinvestigated to different extents. Following the 2 hours infusion, thetissue distribution was high for curcumin in the lung (22.86 ng/g)compared to other tissues (13-254-fold). The next highest tissue was theliver (1.82 ng/g), with distribution in other tissues ranging from0.09-1.14 ng/g. The high distribution of curcumin into the lung may bedue related to fact that it is a very lipophilic compound. A similarpattern was observed for THC following the 2 hour infusion, withcomparable tissue levels of THC to curcumin observed.

Upon 8 hours of infusion, albeit at a lower infusion concentration, theextent of curcumin and THC changed. While the lung and liver again hadthe highest and second highest levels of curcumin, there were clearlyincreased concentrations of curcumin and THC in the liver and lungs with2 hours versus 8 hours levels of 22.86 vs 250.75 ng/g and 1.82 vs 28.38ng/mL, respectively. The highest level in the lung observed, 250.75 ng/gof curcumin translates into a tissue concentration of 0.68 μM acceptingthat 1 gram tissue is equivalent to 1 mL of volume. Curcumin levelsranged from 0.01-2.84 ng/g in other tissues. The levels of THC in thepancreas, kidney and urinary bladder were also increased following 8hours of infusion, while other tissues were comparable to those observedwith the 2 hour infusion. The levels of THC were also increasedfollowing 8 hours of infusion compared to 2 hours infusion. Theincreased tissue incorporation of curcumin in the lung and liver with 8hours of infusion is consistent with the previously reported inabilityto achieve steady-state plasma levels of curcumin during 8 hours ofinfusion, further supporting an enhancement of tissue uptake during thecourse of infusion. A comparison of the tissue partition coefficients(Kp) further support this point and sheds additional light on the impactof short versus longer infusions of curcumin on tissue distribution indogs (Tables 10-11). Firstly, both following 2 and 8 hours of infusion,the majority of the Kp values for curcumin and THC are below one,suggesting a poor tissue distribution of curcuminoids into tissues andconsistent with the low oral bioavailability of curcumin. Low Kp valueshave also been observed in rodent studies and ranged from 0.06-0.25 inthe rat. Exceptions to this are the liver and lung with >1 values of 1.9and 16.8 respectively with 8 hours of infusion. Secondly, the Kp valuesare higher for curcumin than for THC, which to some extent makes sensewith the lower lipophilicity of THC. Thirdly, the Kp values are higheramongst all tissues for both curcumin and THC following the 8 hourinfusion compared to the 2 hour infusion. This latter point highlysupports and enhancement of the tissue distribution of curcuminoids withlonger infusion. In the literature, curcumin has been reported toinhibit the transporter mediated efflux of drugs from cells. At themechanistic level, this may indeed explain the increased uptake ofcurcumin into tissues with a longer infusion and inability to attainsteady-state plasma levels. Essentially, as infusion proceeds, curcuminlevels build-up in tissues and begins to progressively inhibit efflux,resulting in greater tissue sequestration over time, the extent of whichin any one tissue being dependent on the balance between uptake andefflux transporter activity. The higher levels of THC in tissues at 8hours may be a consequence of the metabolism of the higher tissueslevels of curcumin. Thus, in addition to the conclusions reached fromanalysis of the plasma levels of curcumin, the rapid clearance ofcurcumin from the circulation in addition to the impact of the liver andkidney, may also involve a number of tissues and be dependent on theirbalance of transporter mediated uptake and efflux. Curcumin and THC weredistributed amongst all of the tissues investigated with very highlevels compared to other tissues observed in the lung. The liver had thesecond highest levels. With 8 hour infusion, the tissue levels ofcurcumin in the lung and liver increased substantially compared to 2hour infusion, with the pancreas, kidney and urinary bladder alsodisplaying higher tissue levels. Tissue partition coefficients forcurcumin and THC were higher for the 8 hour infusion compared to the 2hour infusion, suggesting that prolonged infusion of curcumin mayfacilitate tissue distribution via a transporter-dependent mechanism.

TABLE 12 Effect of duration of a single dose: 10 mg/kg: 2 hours vs 8hours intravenous curcumin infusion on the ratio of tissue distributionof curcumin: THC in dogs. Concentration of THC A Concentration ofcurcumin 2 h infusion > 8 h B C infusion 8 h infusion > 2 h infusion 8 hinfusion = 2 h infusion lung lung intestinal wall intestinal wall muscleheart heart spleen bladder muscle liver brainstem bladder kidney cortexspleen pancreas hippocampus liver striatum kidney pancreas brainstemcortex striatum hippocampus

As seen in Table 12 above: In column A the THC concentrations are higherin all 13 organs tested following a 2 hour infusion of liposomalcurcumin compared to an 8 hour liposomal curcumin infusion. In column Bthe curcumin concentrations of following intravenous infusions ofliposomal curcumin appear to be both tissue specific and time dependent.The longer infusion (8 hour) distributes preferably to 6 tissues. Incolumn C the curcumin concentrations are not significantly different inthe 2 hour and 8 hour infusions in 7 other tissues. Intertissuevariance. Variance following infusions may be due to several causes:vascular supply, penetration, local tissue clearance/excretion,enzymatic reduction to THC from curcumin.

TABLE 13 Average H₃PO₄ stabilized tissue concentrations (ng/gm) ofcurcumin and THC of 4 dogs 2 male and 2 female following 2 hour and 4dogs following 8 hour infusions of 10 mg/kg liposomal curcumin. Two hourinfusion Eight hour infusion Tissue Curcumin THC Curcumin THC Lung 22.869.37 250.75 6.36 Liver 1.81 4.58 28.38 2.24 Spleen 0.075 1.60 22.90 0.42Pancreas 0.85 0.91 2.84 1.34 Kidney 0.89 4.25 2.76 2.03 Bladder 0.662.25 0.87 1.11 Heart 0.47 0.68 0.74 0.03 Intestinal wall 1.14 2.11 1.110.09 Muscle 0.18 4.19 0.68 2.42 Brainstem 0.45 10.6 0.57 3.02 Cortex0.73 3.08 0.80 0.49 Striatum 0.48 11.11 0.49 3.13 Hippocampus 0.09 6.460.01 1.12

Interpretation. The distribution of intravenous liposomal curcumin tovarious body tissues is not homogeneous, and it appears that the lung,liver and spleen either collect or retain significantly more curcuminthan the remaining tissues. These data show the 8 hour infusion leads tosignificantly higher levels of curcumin in the lung, liver, spleen,pancreas, kidney, and muscle hypothetically due to low enzymaticreduction to THC or decreased clearance. In other tissues: muscle,bladder, heart, and intestinal wall there is no significant difference.Levels of THC are significantly reduced in all tissues receiving the 8hour infusion. These data reflect the net result of the tissue dependentpresence of reductive enzymes, the delivery of curcumin to the tissuesleading to lesser amounts of THC and the pharmacokinetic profile of THC.The brain tissues are remarkably clear for supporting the presence ofTHC over curcumin, in this case prolonged infusion leads to greaterclearance and lesser concentrations. The infusion duration does affectcurcumin and THC metabolism, and may have to be taken into considerationwhen treating different tissue pathologies. For example, cerebraldisorders may be better treated with brief infusions to achieve higherlevels of THC, assuming THC is equally or better effective against brainbased disorders than curcumin.

TABLE 14 H₃PO₄ stabilized vs Non-stabilized tissue analysis following a2 hour infusion of Liposomal curcumin. Curcumin THC +H₃PO₄ −H₃PO₄ +H₃PO₄−H₃PO₄ Lung 22.86 86.80 9.37 17.73 Spleen 0.07 0.48 1.60 1.35 Liver 1.814.28 4.58 2.41 Pancreas 0.85 2.81 0.91 2.01 Brainstem 0.45 0.32 10.602.33 Cortex 0.73 0.52 3.08 0.67 Striatum 0.48 0.32 11.11 6.21Hippocampus 0.09 0.00 6.46 0.75

TABLE 15 H₃PO₄ stabilized vs non-stabilized tissue analysis following an2 hour infusion of Liposomal curcumin. Curcumin THC +H₃PO₄ −H₃PO₄ +H₃PO₄−H₃PO₄ Lung 250.75 317.90 6.36 10.81 Spleen 22.90 28.63 0.42 0.32 Liver28.38 39.38 2.24 2.63 Pancreas 2.84 1.87 1.34 0.33 Brainstem 0.57 0.413.02 0.83 Cortex 0.80 0.72 0.49 0.12 Striatum 0.49 0.14 3.13 1.12Hippocampus 0.01 0.00 1.12 0.01

Following the 2 hour infusion, with regard to curcumin, higher levelswere achieved in the absence of phosphoric acid addition in thefollowing tissues: lung, spleen, liver, pancreas, while higher levels inall brain tissues examined were observed with the addition of phosphoricacid. With regard to THC, all brain, spleen and liver levels were higherwith the addition of phosphoric acid while lung and pancreas tissueswere lower. The patterns in the 8 hour infusions (Table 15) were asfollows: higher levels of curcumin were achieved in the absence ofphosphoric acid in the following tissues: lung, spleen, liver, while theaddition of phosphoric acid induced higher levels in the pancreas. Therewas no significant on the impact of phosphoric acid on curcumin levelsin all brain tissues. Regarding THC levels, the addition of phosphoricacid increased THC levels in all brain, and pancreatic tissues. Theabsence of phosphoric acid addition was associated with higher THClevels in lung tissue, but had no incremental impact in other tissues.

Extrapolating to humans, and based upon the variances in THC formation,and specific tissue levels of curcumin and THC following 8 hour and 2hour infusions of liposomal curcumin including the presence or absenceof added phosphoric acid for stabilization, designing administrationschedules may best be adapted for specific tissue pathologies in orderto achieve optimum therapeutic results. In a 60 kg adult, 370 mg/M2 isequivalent to 10 mg/kg/dose. Converting 10 mg/kg dose in dogs to humans:×0.5=5.0 mg/kg/dose. Clinical applications: decision suggestions foreither 2 hour or 8 hour or longer infusions of liposomal curcumin.

Lung disorders. Curcumin concentrations in the lung are higher in the 8hour infusion, than in the 2 hour infusion, and levels are furtherelevated when analyzed in the presence of phosphoric acid. Curcumin mayhave therapeutic value in treating scleroderma, as it has already beenshown to protect rats from lung fibrosis induced by a variety of agents.THC concentrations in the lung are higher after the 2 hour infusion thanin the 8 hour infusion, and levels are lower when analyzed in thepresence of phosphoric acid. Tetrahydrocurcumin has high anti-oxidantactivity potency in three bioassay models, i.e. the linoleic acidauto-oxidation model, rabbit erythrocyte membrane ghost system, and ratliver microsome system implying that hydrogenation of curcuminoidsincreases anti-oxidant ability.

Liver disorders: Curcumin concentrations in the liver are higher in the8 hour infusion than in the 2 hour infusion, and further elevated in thepresence of phosphoric acid. THC concentrations in the liver are higherafter the 2 hour infusion than the 8 hour infusion, and are increased inthe presence of phosphoric acid. In the 8 hour infusion there was noadvantage to adding phosphoric acid. Spleen disorders: Curcuminconcentrations in the spleen are higher in the 8 hour infusion than inthe 2 hour infusion, and further elevated in the presence of phosphoricacid. Curcumin increases sub G1 cell populations with strongapoptosis-inducing activity. THC concentrations in the spleen are higherafter the 2 hour infusion. Treatment with THC induced autophagic celldeath in human HL-60 promyelocytic leukemia cells by increasingautophage marker acidic vascular organelle formation. Flow cytometryalso confirmed that THC treatment did not increase sub-G1 cellpopulation. Western blot analysis showed that THC significantlydown-regulated phosphatidylinositol 3-kinase/protein kinase B andmitogen-activated protein kinase signalings including decreasing thephosphorylation of mammalian target of rapamycin, glycogen synthasekinase 3Î² and p70 ribosomal protein S6 kinase. Conclusion: these datademonstrated the anticancer efficacy of THC by inducing autophagy, andprovide prevention of human leukemia. Myelofibrosis (MF) a significantdisease burden: 85% of myelofibrosis patients present with splenomegalyand 60% to 80% of MF patients report spleen-related symptoms. In MF,splenomegaly of any degree is clinically relevant, and since themajority of patients with MF experience debilitating symptoms,appropriate treatment should be considered. Muscle disorders: Curcuminconcentrations in muscle tissue are higher in the 8 hour infusion thanin the 2 hour infusion. Pancreatic disorders: Curcumin concentrations inpancreatic disorders are higher in the 8 hour infusion than in the 2hour infusion. THC concentrations are higher after the 2 hour infusionthan after an 8 hour infusion. Kidney disorders: Curcumin concentrationsin renal disorders are higher in the 8 hour infusion than in the 2 hourinfusion. THC concentrations in the kidney are higher after a 2 hourinfusion, than after an 8 hour infusion. Neural disorders: Curcumin inthe Brainstem, Cortex, Striatum, and Hippocampus: either 2 hour or 8hour infusions produce similar concentrations which are unchanged byphosphoric acid addition. Curcumin was effective in reducing amyloidplaque burden, insoluble beta-amyloid peptide.

In the Parkinson's disease model, depletion of dopamine (DA) and DOPAC(3, 4-dihydroxy phenyl acetic acid)) occurs with increased monoamineoxidase (MAO-B) activity. Administration of curcumin (80 mg/kg i.p.) andtetrahydrocurcumin (60 mg/kg i.p.) significantly reversed theMPTP-induced depletion of DA and DOPAC. The MAO-B activity was alsosignificantly inhibited by these compounds. Both curcumin and THC exertneuroprotection against MPTP induced neurotoxicity. THC compared withcurcumin gavage leads to dramatically higher drug plasma levels, howeverresulting brain levels of parent compounds were similar. Levels in theBrainstem, Cortex, Striatum and Hippocampus are increased in the 2 hourinfusion and further increased by phosphoric acid in both 2 hour and 8hour infusions.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method of determining a curcumin level in asample comprising the steps of: providing the sample suspected ofcomprising a curcuminoid; adding a strong acid to the sample, whereinthe strong acid composition is non-buffering; and detecting the amountof curcuminoid in the sample, wherein the non-buffering strong acidreduces the degradation of the curcuminoid in the sample.
 2. The methodof claim 1, wherein the sample is an in vitro sample.
 3. The method ofclaim 1, wherein the sample is an aqueous sample, a supernatant sample,a tears sample, a sputum sample, a blood sample or a bile sample.
 4. Themethod of claim 1, wherein the curcuminoid composition comprisescurcumin and analogues and derivatives selected from curcumin;tetrahydrocurcumin; hexahydrocurcumin and hexahydrocurcuminol; curcuminglucuronide; and curcumin sulfate.
 5. The method of claim 1, wherein thestrong acid is selected from at least one of a phosphoric acid; anorthophosphoric acid; a phosphate salt; or a Na-phosphate.
 6. The methodof claim 1, wherein the curcuminoid composition further comprises aliposome, a phospholipid or a polymer composition to form anencapsulated curcuminoid composition.
 7. The composition of claim 6,wherein the liposome, the phospholipid or the polymer composition isselected from the group consisting of phosphatidylcholine (lecithin),lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine,phosphatidylinositol, sphingomyelin, phosphatidylethanolamine(cephalin), cardiolipin, phosphatidic acid, cerebrosides,dicetylphosphate, phosphatidylcholine, anddipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine,hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid,fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, anddiacylglycerolsuccinate; or wherein the polymer composition is selectedfrom the group consisting of polyesters, polylactides, polyglycolides,polycaprolactones, polyanhydrides, polyamides, polyurethanes,polyesteramides, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoesters,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), copolymers, terpolymers, and combinations or mixturesthereof.
 8. The composition of claim 6, wherein the encapsulatedcurcuminoid composition has a size of about 10-900 nm.
 9. A kit fordetecting curcumin comprising: a first vial for a biological sample, anda second vial with an amount of a strong acid that is a non-bufferingsufficient to stabilize a curcuminoid in a biological sample; and a setof instructions for stabilizing a curcumin sample using thenon-buffering phosphate composition, wherein the non-buffering phosphatecomposition comprises a phosphoric acid; an orthophosphoric acid; aphosphate salt; or a Na-phosphate to stabilize curcumin;tetrahydrocurcumin; hexahydrocurcumin and hexahydrocurcuminol; curcuminglucuronide; and curcumin sulfate.
 10. A method of stabilizingstabilizing a curcumin composition in plasma sample or a bile sampleagainst degradation during an analytical processes comprising the stepsof: providing a sample comprising a curcumin composition, wherein thesample is a bile sample or a blood sample and the curcumin compositionis selected from curcumin; tetrahydrocurcumin; hexahydrocurcumin andhexahydrocurcuminol; curcumin glucuronide; and curcumin sulfate; addinga phosphate composition to the sample, wherein the phosphate compositionis non-buffering and is selected from a phosphoric acid; anorthophosphoric acid; a phosphate salt; or a Na-phosphate; and detectingthe amount of curcuminoid in the sample, wherein the non-bufferingphosphate composition reduces the degradation of the curcuminoid in thesample.
 11. A stabilized curcumin composition comprising: a curcumincomposition and a phosphate composition, wherein the phosphatecomposition is non-buffering and is provided in an amount sufficient toat least one of reduce the degradation or stabilize the curcumin in asample.
 12. The stabilized curcumin composition of claim 14, wherein thecurcumin composition selected from Curcumin; tetrahydrocurcumin;hexahydrocurcumin and hexahydrocurcuminol; curcumin glucuronide; andcurcumin sulfate.
 13. The stabilized curcumin composition of claim 14,wherein the phosphate composition is selected from at least one of aphosphoric acid, a orthophosphoric acid, a phosphate salt, or aNa-phosphate.
 14. The stabilized curcumin composition of claim 14,further comprising a liposome to form a liposomal curcumin composition.15. The stabilized curcumin composition of claim 14, wherein thestabilized curcumin composition comprises a solution dosage form.
 16. Amethod of performing a clinical trial to evaluate a candidate drugcomprising a curcumin or curcuminoid believed to be useful in treating amedical condition, the method comprising: (a) obtaining a first tissuesamples prior to providing the candidate substance from tissue suspectedfrom a set of patients; (b) administering the candidate drug to a firstsubset of the patients, and a placebo to a second subset of thepatients; (c) repeating step (a) after the administration of thecandidate drug or the placebo; and (d) obtaining a second tissue samplefrom the first and second set of patients and stabilizing the curcuminor curcuminoids in the second tissue samples by adding an effectiveamount of a non-buffering phosphate; and (e) determining of there is astatistically significant difference in the amount of curcumin orcurcuminoids in the second tissue samples between the first and secondsubset of patients, wherein a statistically significant reductionindicates that the candidate drug is useful in treating said diseasestate.
 17. The stabilized curcumin composition of claim 19, wherein thecurcumin composition selected from at least one of curcumin;tetrahydrocurcumin; hexahydrocurcumin and hexahydrocurcuminol; curcuminglucuronide; or curcumin sulfate.